The goal of this project is to determine the mechanism by which cellular signaling pathways target chromatin with a particular focus on signaling mediated through cyclic nucleotides. At gene promoters targeted by various signaling pathways, histones are often found to be modified in response to activation of these pathways. The changes in histone modification influence the activity of these promoters in a variety of ways. In addition, some histone modifications in bulk cellular chromatin are found to be cell cycle dependent. For example, chromatin-associated histone H3 becomes phosphorylated just prior to mitosis and is then dephosphorylated upon exit from mitosis. The signaling cascades which lead to both gene-specific and bulk changes in histone modifications are still largely unknown.Our previous work has shown that cAMP signaling causes repression of the MMTV promoter in a glucocorticoid-independent fashion only when it is incorporated into ordered, replicating chromatin in cultured cells. In contrast, a transiently-transfected MMTV promoter construct, which does not have an organized nucleoprotein structure, is activated by cAMP signaling. These results emphasize the importance of chromatin structure in understanding transcriptional regulation of genes targeted by signaling pathways. We have determined thatthe cAMP-induced repression of the MMTV promoter in organized chromatin involves deacetylation and dephosphorylation of histone H3 in a restricted area of the promoter. These changes are particularly pronounced in a region of the promoter known to contain negative regulatory elements. These results indicate the cAMP-dependent recruitment of corepressor complexes containing chromatin modifying activities. In contrast to transcriptional activation by cAMP signaling, very little is understood about the mechanisms by which this pathway induces transcriptional repression. Our current efforts are directed towards identifying the sequences necessary for repression and the nature of the repressor complexes involved.In the process of studying histone modification at the MMTV promoter in response to signaling pathways, we determined that cAMP signaling causes a drastic reduction in bulk levels of histone H3 phosphorylation in our breast adenocarcinoma cell lines. Since this particular modification has been associated with both transcriptional activity and proper progression through mitosis, we are very interested in understanding the mechanism of this dephosphorylation and its effects on cell biology. We have determined the sites of dephosphorylation on histone H3 as being those involved in the above processes. Interestingly, the cAMP-induced dephosphorylation is not mediated through the PKA- or Epac-dependent pathways, indicating a unique mode of cAMP-signaling. Our studies also indicate that okadaic acid sensitive phosphatases are involved in H3 dephosphorylation. Currently, we are focused on characterizing the cAMP signaling cascade which mediates H3 dephosphorylation and examining its ultimate effects on cell cycle progression.